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Related Experiment Videos

A novel highly reproducible quantitative competitve RT PCR system

J Zhang1, C D Byrne

  • 1Department of Clinical Biochemistry , University of Cambridge, Cambridge CB2 2QR, UK.

Journal of Molecular Biology
|January 31, 1998
PubMed
Summary

A new quantitative competitive reverse transcriptase (RT) PCR method improves precision for measuring fibrinogen mRNA levels. This technique accurately detects reductions in fibrinogen gene expression linked to dietary changes.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Plasma fibrinogen concentration changes are clinically significant.
  • Reverse transcriptase PCR (RT-PCR) imprecision can arise from altered template ratios during cDNA synthesis.

Purpose of the Study:

  • To develop a novel quantitative competitive RT-PCR system for precise measurement of fibrinogen mRNA levels.
  • To improve the reproducibility of RT-PCR assays.

Main Methods:

  • A quantitative competitive RT-PCR system was designed involving RNA titration, multi-cycle amplification, and quality control reactions.
  • Three criteria for RT-PCR data analysis were proposed to enhance reproducibility.

Main Results:

  • The developed RT-PCR system demonstrated improved reproducibility with an inter-assay coefficient of variation (CV) of 6.5%.

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  • The system detected significant reductions in fibrinogen alpha, beta, and gamma mRNA levels in rats subjected to dietary protein restriction.
  • A parallel decrease in plasma fibrinogen concentration was observed.

    • Conclusions:

    • The novel quantitative competitive RT-PCR system offers a precise and reproducible method for assessing fibrinogen mRNA expression.
    • Dietary protein restriction in weaned rats leads to decreased fibrinogen gene expression and plasma concentrations.